Diabetes is characterized by impaired insulin secretion from pancreatic β-cells, insulin resistance or both (Cavaghan, M. K., et al., J. Clin. Invest. 2000, 106, 329). Majority of type 2 diabetic patients can be treated with agents that reduces hepatic glucose production (glucagon antagonist), reduce glucose absorption form GIT, stimulate β-cell function (insulin secretagogues) or with agents that enhance the tissue sensitivity of the patients towards insulin (insulin sensitizes). The drugs presently used to treat type 2 diabetes include α-glucosidase inhibitors, insulin sensitizers, insulin secretagogues and KATP channel blocker (Chehade, J. M., et al., Drugs, 2000, 60, 95). However, almost one-half of type 2 diabetic subjects lose their response to these agents, over a period of time and thereby require insulin therapy. Insulin treatment has several drawbacks, it is injectable, produces hypoglycemia and causes weight gain (Burge, M. R., Diabetes Obes. Metab., 1999, 1, 199).
Problems with the current treatment necessitate new therapies to treat type 2 diabetes. In this regard, glucagon-like peptide-1 (GLP-1) agonist, which promote glucose-dependent insulin secretion in the pancreas and glucagon receptor antagonist, which inhibit hepatic glucose production by inhibiting glycogenolysis and gluconeogenesis, were found to be therapeutically potential. Thus GLP-1 agonist and glucagon antagonist together were found to reduce the circulating glucose levels and represent useful therapeutic agents for the treatment and prevention of type 2 diabetes (Perry, T. A., et al., Trends Pharmacol. Sci., 2003, 24, 377).
Glucagon and GLP-1 are members of structurally related peptide hormone family (secretin family). Glucagon and GLP-1 constitute a highly homologous set of peptides because these two hormones originate from a common precursor, preproglucagon, which upon tissue-specific processing leads to production of GLP-1 predominantly in the intestine and glucagon in the pancreas (Jiang, G., et al., Am. J. Physiol. Endocrinol. Metab., 2003, 284, E671-678). The receptors for these two peptides are homologous (58% identity) and belong to the class B family of G-protein coupled receptors (GPCRs). Class-B GPCRs is also called as the secretin receptor family, which consist of 15 peptide-binding receptors in humans. GPCR receptors comprise an extracellular N-terminal domain of 100-160 residues, connected to a juxtamembrane domain (J-domain) of seven membrane-spanning α-helices with intervening loops and a C-terminal tail (Brubaker, P. L., et al., Receptors Channels, 2002, 8, 179). Class B GPCRs are activated by endogenous peptide ligands of intermediate size, typically 30-40 amino acids (Hoare, S. R. J., Drug. Discovery Today, 2005, 10, 423; Gether, U., Endocrine Reviews, 2000, 21, 90).
Glucagon is a 29-amino acid peptide hormone processed from proglucagon in pancreatic α-cells by PC2. Glucagon acts via a seven transmembrane GPCRs, consisting of 485 amino acids. Glucagon is released into the bloodstream when circulating glucose is low. The main physiological role of glucagon is to stimulate hepatic glucose output, thereby leading to increase in glycemia (Tan, K., et al., Diabetologia, 1985, 28, 435). Glucagon provides the major counterregulatory mechanism for insulin in maintaining glucose homeostasis in vivo. Glucagon and its receptor represent potential targets for the treatment of diabetes. Antagonising glucagon action by blocking the action of the secreted glucagon at glucagon receptor (glucagon antagonist) or by inhibiting (suppressing) the glucagon production itself represents a new avenue for intervention of diabetes and metabolic disorders (Unson, C. G., et al., Peptides, 1989, 10, 1171; Parker, J. C., Diabetes, 2000, 49, 2079; Johnson, D. G., Science, 1982, 215, 1115; Ahn, J. M., JMC, 2001, 44(9), 1372-1379).
The GLP-1 (7-36) amide is a product of the preproglucagon gene, which is secreted from intestinal L-cells, in response to the ingestion of food. The physiological action of GLP-1 has gained considerable interest. GLP-1 exerts multiple action by stimulating insulin secretion from pancreatic β-cells, in a glucose dependent manner (insulinotropic action). GLP-1 lowers circulating plasma glucagon concentration, by inhibiting its secretion (production) from α-cells (Drucker D. J., Endocrinology, 2001, 142, 521-527). GLP-1 also exhibits properties like stimulation of β-cell growth, appetite suppression, delayed gastric emptying and stimulation of insulin sensitivity (Nauck, M. A., Horm. Metab. Res., 2004, 36, 852). Currently, various analogs of GLP-1 and EX-4, such as Liraglutide/NN2211 (Novo Nordisk; Phase-III; WO 1998 008871), BIM 51077 (Ipsen; Phase-II; WO 2000 034331), CJC-1131 (ConjuChem; Phase-II; WO 2000 069911) and ZP-10 (Zealand and Aventis; Phase-II; WO 2001 004156) are in different stages of clinical development (Nauck M. A., Regulatory Peptides, 2004, 115, 13). Recently, BYETTA® (Exendin-4, AC 2933; U.S. Pat. No. 5,424,286), has been launched in the US market (Amylin and Lilly). However, all the existing GLP-1 agonists are delivered by the parenteral route of administration, so the patient incompliance is major problem with the existing GLP-1 based therapy.
The effector system of glucagon and GLP-1 receptors is the Adenylyl Cyclase (AC) enzyme. Interaction of glucagon or GLP-1 agonist with glucagon or GLP-1 receptors (GLP-1 R) respectively causes activation of AC, which converts ATP to cAMP. Increase in the intracellular cAMP level raises the ratio of ADP/ATP, thereby initiating the cell depolarization (due to closure of KATP channel). Increase in the intracellular cAMP level also activates Protein Kinase (PK-A and PK-C), which raises the cystolic Ca2+ concentration, by opening of L-type of Ca2+ channel. An increase in the intracellular Ca2+ leads to exocytosis of insulin, in pancreatic β-cells and glucagon peptide in α-cells (Fehmann, H. C., Endocr. Rev., 1995, 16, 390).
GLP-1 and glucagon sequences alignment shown below represent the primary structural relationships:
Glucagon:(Seq. ID No: 1)NH2-1HSQGTFTSD9YSKYLDSRRAQDFVQWLMNT29-CONH2 GLP-1(7-36):(Seq. ID No: 2)NH2-1HAEGTFTSD9VSSYLEGQAAKEFIAWLVKGR30-CONH2
First N-terminal 1-9 residues of GLP-1 peptide, with C-terminal amide: NH3(+)-1HAE(−)GTFTSD9(−)-CONH2 (Seq. ID No: 3): Net charge Negative
First N-terminal 1-9 residues of Glucagon peptide, with C-terminal amide: NH3(+)-1HSQGTFTSD9(−)-CONH2 (Seq. ID No: 4): Net charge Neutral
Single-letter abbreviations for amino acids can be found in Zubay, G., Biochemistry 2nd ed., 1988, MacMillan Publishing, New York, p. 33.
Native or synthetic GLP-1 peptides are rapidly metabolized by the proteolytic enzymes, such as dipeptidyl peptidase-IV (DPP-IV) into an inactive metabolite, thereby limiting the use of GLP-1 as a drug (Deacon, C. F., Regulatory Peptides, 2005, 128, 117). Similarly, several nonpeptidyl and peptidyl glucagon receptor antagonist of diverse structures have been reported over recent years, but none of them are in active development or under clinical trials (Kurukulasuriya, R., Expert Opinion Therapeutic Patents, 2005, 15, 1739; Lau, J., J. Med. Chem., 2007, 50, 113; Petersen, K. F. Diabetologia, 2001, 44, 2018; Cascieri, M. A., JBC, 1999, 274, 8694). It is believed that identifying nonpeptide ligands (especially agonist) for class B GPCRs is the principle bottleneck in drug discovery. HTS has apparently yielded few hits (US 2005/6927214; WO 2000/042026; US 2007/0043093), however, screening of those hits against corresponding receptors, especially under in vivo condition (animal models) prone to be false negatives (Murphy, K. G., PNAS, 2007, 104, 689).
Glucagon and GLP-1 both play major roles in overall glucose homeostasis (Drucker, D. J., J. Clin. Invest., 2007, 117, 24; Bollyky, J., J. Clin. Endocrinol. Metab., 2007, 92, 2879). Glucagon increases plasma glucose concentrations by stimulating gluconeogenesis and glycogenolysis in the liver while GLP-1 lowers plasma glucose concentrations mediated by glucose dependent insulin secretion (Mojsov, S., et al., JBC., 1990, 265, 8001). Knowing the importance of both glucagon peptide and GLP-1 in maintaining normal blood glucose concentrations, in the recent years, there has been considerable interest in identifying a single ligand, which act as glucagon receptor antagonists and GLP-1 receptor agonists (Claus, T. H., J. Endocrinology, 2007, 192, 371; Pan C. Q., JBC, 2006, 281, 12506).
Although identification of potent nonpeptide GLP-1 agonist may be difficult (Chen, D., PNAS, 2007, 104, 943; Knudsen, L. B., PNAS, 2007, 104, 937) but the design of a hybrid peptidomimetic acting as both glucagon antagonist and GLP-1 receptor agonist would likely to provide a novel approach for the treatment of type 2 diabetes (Claus, T. H., J. Endocrinology, 2007, 192, 371). Recently, series of chimeric peptides has been reported, which act as both GLP-1 receptor agonist and glucagon receptor antagonist, constructed mainly by combining the N-terminal residues of glucagon peptide (residues 1-26) with last C-terminal 4 residues of GLP-1 peptide (VKGR) (Pan C. Q., et al., U.S. Pat. No. 6,864,069 B2; Pan C. Q., JBC, 2006, 281, 12506).
Structure-activity relationship (SAR) studies have been reported in the literature to determine the role of individual amino acids in both the glucagon and GLP-1 sequences (Runge, S., JBC, 2003, 278, 28005; Mann, R., Biochem. Soc. Trans., 2007, 35, 713). Glucagon and GLP-1 have no defined structure in aqueous solution, but in the presence of micelles or in the membrane mimetic environment, they adopt an alpha-helical structure in the mid-section, with flexible N- and C-terminal regions (Thornton, K., Biochemistry, 1994, 33, 3532; Neidigh, J. W., Biochemistry, 2001, 40, 13188). This suggests that the helical structure is required for binding of peptide ligands to their respective receptors. Mutations or deletion of amino acids in the N-terminal region of both the peptides results in receptor antagonists or inactive compounds, suggesting the importance of the N-terminus for receptor activation by both the glucagon and GLP-1 peptides (Hjorth, S. A., JBC., 1994, 269, 30121; Green, B. D., J. Mol. Endocrinology, 2003, 31, 529). In vivo, GLP-1 gets rapidly degraded by dipeptidyl-peptidase IV (DPP IV), a protease responsible for cleaving peptides containing proline or alanine residues in the penultimate N-terminal position, resulting in the inactive metabolites. Substitution of the DPP-IV susceptible sites, such as substitution of Ala at 2nd position of GLP-1 peptide with D-Ala, Aib or Hfl (hexafluoroleucine) greatly improves plasma stability (Deacon, C. F., Diabetes, 1998, 47, 764; Meng, H., J. Med. Chem., 2008, 51, 7303-7307).
In the present investigation, we found that coupling of N-terminal sequence of glucagon peptide (first 1-9 residues, Seq. ID. No. 4) with a dipeptide of two unnatural amino acids resulted in the identification of novel class of peptidomimetics having both the glucagon antagonistic and GLP-1 agonistic activities, at varying degree of selectivity. To enhance the duration of action and stability against DPP-IV enzyme, we have site-specifically modified the hybrid peptidomimetics selectively at position Z2 with unnatural amino acids such as D-Ala, Aib, α-methyl proline (α-Me-Pro), 1-amino-cyclopentanecarboxylic acid (APP) and 1-amino-cyclopropane carboxylic acid (ACP) and succeeded in identifying short peptidomimetics. Some of the peptidomimetics showed efficacy even by oral route of administration, while retaining both the glucagon antagonistic and GLP-1 agonistic activities.